1. Introduction 6
2. Background of the Invention 6
2.1. Hematopoietic Stem and Progenitor Cells 6
2.2. Reconstitution of the Hematopoietic System 9
2.3. Cryopreservation of Cells 13
2.4. Gene Therapy 14
3. Summary of the Invention 16
3.1. Definitions 16
4. Description of the Figures 19
5. Detailed Description of the Invention 20
5.1. Isolation of Fetal or Neonatal Hematopoietic Stem and Progenitor Cells 25
5.1.1. Collection of Neonatal Blood 26
5.1.1.1. Volume 26
5.1.1.2. Preferred Aspects 28
5.1.1.2.1. Collection Kit 28
5.1.1.2.2. Vaginal Delivery of the Term Infant 29
5.1.1.2.3. Other Circumstances of Birth and Delivery 30
xe2x80x835.1.1.2.3.1. Premature Birth 30
xe2x80x835.1.1.2.3.2. Multiple Births 30
xe2x80x835.1.1.2.3.3. Caesarian Delivery 31
xe2x80x835.1.1.2.3.4. Complicated Delivery 31
xe2x80x835.1.1.2.3.5. Abnormal Placenta 32
xe2x80x835.1.1.2.3.6. Collection from the Delivered Placenta 32
xe2x80x835.1.1.2.3.7. Medical Conditions of the Mother 32
xe2x80x835.1.1.2.3.8. Unplanned Delivery 33
5.1.1.2.4. Recordation of Data 33
5.1.2. Inspection and Testing of Neonatal Blood 34
5.1.3. Optional Procedures 36
5.1.3.1. Enrichment for Hematopoietic Stem and Progenitor Cells: Cell Separation Procedures 37
5.1.3.2. In Vitro Cultures of Hematopoietic Stem and Progenitor Cells 43
5.2. Cryopreservation 43
5.3. Recovering Stem and Progenitor Cells from the Frozen State 47
5.3.1. Thawing 47
5.3.2. Optional Procedures 47
5.4. Examination of Cells Recovered for Clinical Therapy 48
5.4.1. Identity Testing 49
5.4.2. Assays for Stem and Progenitor Cells 49
5.5. Hematopoietic Reconstitution 50
5.6. Therapeutic Uses 51
5.6.1. Diseases Resulting from a Failure or Dysfunction of Normal Blood Cell Production and Maturation 56
5.6.2. Hematopoietic Malignancies 59
5.6.3. Malignant Solid Tumors of Non-Hematopoietic Origin 59
5.6.4. Autoimmune Disorders 60
5.6.5. Gene Therapy 61
5.6.6. Miscellaneous Disorders Involving Immune Mechanisms 64
5.7 Generation and Use of Hematopoietic Stem and Progenitor Cell Progeny 65
6. Examples 66
6.1. Collection of Human Umbilical Cord Blood and Placental Blood 66
6.2. Hematopoietic Stem and Progenitor Cells in Collected Cord Blood 68
6.3. Enrichment for Human Hematopoietic Stem and Progenitor Cells: Cell Separation Procedures 75
6.3.1. Density Separations 76
6.3.2. Adherence/Non-Adherence Separation 80
6.4. Cryopreservation of Cord Blood Stem and Progenitor Cells 81
6.5. Cell Thawing 82
6.6. Human Hematopoietic Stem and Progenitor Cell Assays 83
6.6.1. CFU-GM Assay 84
6.6.1.1. Preparation of McCoy""s 5A Medium 86
6.6.1.2. Preparation of Human 5637 Urinary Bladder Carcinoma Cell Line Conditioned Medium 87
6.6.1.3. Preparation of Murine Pokeweed Mitogen Spleen Cell Conditioned Medium 88
6.6.2. BFU-E-2 and BFU-E-1/CFU-GEMM Assay 89
6.6.2.1. Preparation of 2.1% Methyl Cellulose 91
6.6.2.2 Preparation of Hemin 92
6.6.2.3. Preparation of Iscove""s Modified Dulbecco""s Medium 92
6.6.3. Stem Cell Colony Forming Unit Assay 93
6.6.4. Assay of the Proliferative Status of Stem and Progenitor Cells 93
6.7. Recovery After Freeze-Thawing of Human Hematopoietic Progenitor Cells Derived from Cord Blood 95
6.8. Calculations of the Reconstituting Potential of Cord Blood 98
6.9. In Vitro Culture Conditions for Hematopoietic Stem and Progenitor Cells 100
6.10. Mouse Dissection Protocols 100
6.10.1. Bone Marrow Dissection 101
6.10.2. Spleen Dissection 102
6.11. Hematopoietic Reconstitution of Adult Mice with Syngeneic Fetal or Neonatal Stem Cells 103
6.11.1. Hematopoietic Reconstitution of Lethally-Irradiated Mice with Stem Cells in Blood of the Near-Term Fetus 103
6.11.2. Hematopoietic Reconstitution of Mice with a Lesser Volume of Near-Term Fetal Blood But Not with Adult Blood 105
6.11.3. Hematopoietic Reconstitution with Blood of Newborn Mice in Volumes as Low as Ten Microliters 108
6.11.4. Hematopoietic Reconstitution with Blood of Newborn Mice in Volumes of 10 or 15 Microliters 110
6.12 Hematopoietic Reconstitution For Treatment of Fanconi""s Anemia 111
6.13. Flowchart: Description of a Service 114
The present invention is directed to hematopoietic stem and progenitor cells of neonatal or fetal blood, that are cryopreserved, and the therapeutic uses of such stem and progenitor cells upon thawing. Such cells can be therapeutically valuable for hematopoietic reconstitution in patients with various diseases and disorders. In a preferred embodiment, neonatal cells that have been cryopreserved and thawed, can be used for autologous (self) hematopoietic reconstitution.
The invention also relates to methods for collection and cryopreservation of the neonatal and fetal stem and progenitor cells of the invention.
The morphologically recognizable and functionally capable cells circulating in blood include erythrocytes, neutrophilic, eosinophilic, and basophilic granulocytes, B-, T-, nonB-, non T-lymphocytes, and platelets. These mature cells derive from and are replaced, on demand, by morphologically recognizable dividing precursor cells for the respective lineages such as erythroblasts for the erythrocyte series, myeloblasts, promyelocytes and myelocytes for the granulocyte series, and megakaryocytes for the platelets. The precursor cells derive from more primitive cells that can simplistically be divided into two major subgroups: stem cells and progenitor cells (for review, see Broxmeyer, H. E., 1983, xe2x80x9cColony Assays of Hematopoietic Progenitor Cells and Correlations to Clinical Situations,xe2x80x9d CRC Critical Reviews in Oncology/Hematology 1(3):227-257). The definitions of stem and progenitor cells are operational and depend on functional, rather than on morphological, criteria. Stem cells have extensive self-renewal or self-maintenance capacity (Lajtha, L. G., 1979, Differentiation 14:23), a necessity since absence or depletion of these cells could result in the complete depletion of one or more cell lineages, events that would lead within a short time to disease and death. Some of one stem cells differentiate upon need, but some stem cells or their daughter cells produce other stem cells to maintain the precious pool of these cells. Thus, in addition to maintaining their own kind, pluripotential stem cells are capable of differentiation into several sublines of progenitor cells with more limited self-renewal capacity or no self-renewal capacity. These progenitor cells ultimately give rise to the morphologically recognizable precursor cells. The progenitor cells are capable of proliferating and differentiating along one, or more than one, of the myeloid differentiation pathways (Lajtha, L. G. (Rapporteur), 1979, Blood Cells 5:447).
Stem and progenitor cells make up a very small percentage of the nucleated cells in the bone marrow, spleen, and blood. About ten times fewer of these cells are present in the spleen relative to the bone marrow, with even less present in the adult blood. As an example, approximately one in one thousand nucleated bone marrow cells is a progenitor cell; stem cells occur at a lower frequency. These progenitor and stem cells have been detected and assayed for by placing dispersed suspensions of these cells into irradiated mice, and noting those cells that seeded to an organ such as the spleen and which found the environment conducive to proliferation and differentiation. These cells have also been quantified by immobilizing the cells outside of the body in culture plates (in vitro) in a semi-solid support medium such as agar, methylcellulose, or plasma clot in the presence of culture medium and certain defined biomolecules or cell populations which produce and release these molecules. Under the appropriate growth conditions, the stem or progenitor cells will go through a catenated sequence of proliferation and differentiation yielding mature end stage progeny, which thus allows the determination of the cell type giving rise to the colony. If the colony contains granulocytes, macrophages, erythrocytes, and megakaryocytes (the precursors to platelets, then the cell giving rise to them would have been a pluripotential cell. To determine if these cells have self-renewal capacities, or stemness, and can thus produce more of their own kind, cells from these colonies can be replated in vivo or in vitro. Those colonies, which upon replating into secondary culture plates, give rise to more colonies containing cells of multilineages, would have contained cells with some degree of stemness. The stem cell and progenitor cell compartments are themselves heterogeneous with varying degrees of self-renewal or proliferative capacities. A model of the stem cell compartment has been proposed based on the functional capacities of the cell (Hellman, S., et al., 1983, J. Clin. Oncol. 1:227-284). Self-renewal would appear to be greater in those stem cells with the shortest history of cell division, and this self-renewal would become progressively more limited with subsequent division of the cells.
A human hematopoietic colony-forming cell with the ability to generate progenitors for secondary colonies has been identified in human umbilical cord blood (Nakahata, T. and Ogawa, M., 1982, J. Clin. Invest. 70:1324-1328). In addition, hematopoietic stem cells have been demonstrated in human umbilical cord blood, by colony formation, to occur at a much higher level than that found in the adult (Prindull, G., et al., 1978, Acta Paediatr. Scand. 67:413-416; Knudtzon, S., 1974, Blood 43(3):357-361). The presence of circulating hematopoietic progenitor cells in human fetal blood (Linch, D. C., et al., 1982, Blood 59(5):976-979) and in cord blood (Fauser, A. A. and Messner, H. A., 1978, Blood 52(6):1243-1248) has also been shown. Human fetal and neonatal blood has been reported to contain megakaryocyte and burst erythroblast progenitors (Vainchenker, W., et al., 1979, Blood Cells 5:15-42), with increased numbers of erythroid progenitors in human cord blood or fetal liver relative to adult blood (Hassan, M. W., et al., 1979, Br. J. Haematol. 41:477-484; Tchernia, G., et 1., 1981, J. Lab. Clin. Med. 97(3):322-331). Studies have suggested some differences between cord blood and bone marrow cells in the characteristics of CFU-GM (colony forming unit-granulocyte, macrophage) which express surface Ia antigens (Koizumi, S., et al., 1982, Blood 60(4):1046-1049).
U.S. Pat. No. 4,714,680 discloses cell suspensions comprising human stem and progenitor cells and methods for isolating such suspensions, and the use of the cell suspensions for hematopoietic reconstitution.
Reconstitution of the hematopoietic system has been accomplished by bone marrow transplantation. Lorenz and coworkers showed that mice could be protected against lethal irradiation by intravenous infusion of bone marrow (Lorenz, E., et al., 1951, J. Natl. Cancer Inst. 12:197-201). Later research demonstrated that the protection resulted from colonization of recipient bone marrow by the infused cells (Lindsley, D. L., et al., 1955, Proc. Soc. Exp. Biol. Med. 90:512-515; Nowell, P. C., et al., 1956, Cancer Res. 16:258-261; Mitchison, N. A., 1956, Br. J. Exp. Pathol. 37:239-247; Thomas, E. D., et al., 1957, N. Engl. J. Med. 257:491-496). Thus, stem and progenitor cells in donated bone marrow can multiply and replace the blood cells responsible for protective immunity, tissue repair, clotting, and other functions of the blood. In a successful bone marrow transplantation, the blood, bone marrow, spleen, thymus and other organs of immunity are repopulated with cells derived from the donor.
U.S. Pat. No. 4,721,096 by Naughton et al. discloses a method of hematopoietic reconstitution which comprises obtaining and cryopreserving bone marrow, replicating the bone marrow cells in vitro, and then infusing the cells into patient.
Bone marrow has been used with increasing success to treat various fatal or crippling diseases, including certain types of anemias such as aplastic anemia (Thomas, E. D., et al., Feb. 5, 1972, The Lancet, pp. 284-289), Fanconi""s anemia (Gluckman, E., et al., 1980, Brit. J. Haematol. 45:557-564; Gluckman, E., et al., 1983, Brit. J. Haematol. 54:431-440; Gluckman, E., et al., 1984, Seminars in Hematology:21 (1):20-26), immune deficiencies (Good, R. A., et al., 1985, Cellular Immunol. 82:36-54), cancers such as lymphomas or leukemias (Cahn, J. Y., et al., 1986, Brit. J. Haematol. 63:457-470; Blume, K. J. and Forman, S. J., 1982, J. Cell. Physiol. Supp. 1:99-102; Cheever, M. A., et al., 1982, N. Engl. J. Med. 307(8):479-481), carcinomas (Blijham, G., et al., 1981, Eur. J. Cancer 17(4):433-441), various solid tumors (Ekert, H., et al., 1982, Cancer 49:603-609; Spitzer, G., et al., 1980, Cancer 45:3075-3085), and genetic disorders of hematopoiesis. Bone marrow transplantation has also recently been applied to the treatment of inherited storage diseases (Hobbs, J. R., 1981, Lancet 2:735-739), thalassemia major (Thomas, E. D., et al., 1982, Lancet 2:227-229), sickle cell disease (Johnson, F. J., et al., 1984, N. Engl. J. Med. 311:780-783), and osteopetrosis (Coccia, P. F., et al., 1980, N. Engl. J. Med. 302:701-708) (for general discussions, see Storb, R. and Thomas, E. D., 1983, Immunol. Rev. 71:77-102; O""Reilly, R., et al., 1984, Sem. Hematol. 21(3):188-221; 1969, Bone-Marrow Conservation, Culture and Transplantation, Proceedings of a Panel, Moscow, Jul. 22-26, 1968, International Atomic Energy Agency, Vienna; McGlave, P. B., et al., 1985, in Recent Advances in Haematology, Hoffbrand, A. V., ed., Churchill Livingstone, London, pp. 171-197).
Present use of bone marrow transplantation is severely restricted, since it is extremely rare to have perfectly matched (genetically identical) donors, except in cases where an identical twin is available or where bone marrow cells of a patient in remission are stored in a viable frozen state. Even in such an autologous system, the danger due to undetectable contamination with malignant cells, and the necessity of having a patient healthy enough to undergo marrow procurement, present serious limitations. (For reviews of autologous bone marrow transplantation, see Herzig, R. H., 1983, in Bone Marrow Transplantation, Weiner, R. S., et al., eds., The Committee On Technical Workshops, American Association of Blood Banks, Arlington, Va.; Dicke, K. A., et al., 1984, Sem. Hematol. 21(2):109-122; Spitzer, G., et al., 1984, Cancer 54 (September 15 Suppl.):1216-1225). Except in such autologous cases, there is an inevitable genetic mismatch of some degree, which entails serious and sometimes lethal complications. These complications are two-fold. First, the patient is usually immunologically incapacited by drugs beforehand, in order to avoid immune rejection of the foreign bone marrow cells (host versus graft reaction). Second, when and if the donated bone marrow cells become established, they can attack the patient (graft versus host disease), who is recognized as foreign. Even with closely matched family donors, these complications of partial mismatching are the cause of substantial mortality and morbidity directly due to bone marrow transplantation from a genetically different individual.
Peripheral blood has also been investigated as a source of stem cells for hematopoietic reconstitution (Nothdurtt, W., et al., 1977, Scand. J. Haematol. 19:470-481; Sarpel, S. C., et al., 1979, Exp. Hematol. 7:113-120; Ragharachar, A., et al., 1983, J. Cell. Biochem. Suppl. 7A:78; Juttner, C. A., et al., 1985, Brit. J. Haematol. 61:739-745; Abrams, R. A., et al., 1983, J. Cell. Biochem. Suppl. 7A:53; Prummer, O., et al., 1985, Exp. Hematol. 13:891-898). In some studies, promising results have been obtained for patients with various leukemias (Reiffers, J., et al., 1986, Exp. Hematol. 14:312-315 (using cryopreserved cells); Goldman, J. M., et al., 1980, Br. J. Haematol. 45:223-231; Ti .ly, H., et al., Jul. 19, 1986, The Lancet, pp. 154-155; see also To, L. B. and Juttner, C. A., 1987, Brit. J. Haematol. 66: 285-288, and references cited therein); and with lymphoma (Korbling, M., et al., 1986, Blood 67:529-532). It has been implied that the ability of autologous peripheral adult blood to reconstitute the hematopoietic system, seen in some cancer patients, is associated with the far greater numbers of circulating progenitor cells in the peripheral blood produced after cytoreduction due to intensive chemotherapy and/or irradiation (the rebound phenomenon) (To, L. B. and Juttner, C. A., 1987, Annot., Brit. J. Haematol. 66:285-288; see also 1987, Brit. J. Haematol. 67:252-253, and references cited therein). Other studies using peripheral blood have failed to effect reconstitution (Hershko, C., et al., 1979, The Lancet 1:945-947; Ochs, H. D., et al., 1981, Pediatr. Res. 15(4 Part 2):601).
Studies have also investigated the use of fetal liver cell transplantation (Cain, G. R., et al., 1986, Transplantation 41(1):32-25; Ochs, H. D., et al., 1981, Pediatr. Res. 15(4 part 2):601; Paige, C. J., et al., 1981, J. Exp. Med. 153:154-165; Touraine, J. L., 1980, Excerpta Med. 514:277; Touraine, J. L., 1983, Birth Defects 19:139; see also Good, R. A., et al., 1983, Cellular Immunol. 82:44-45 and references cited therein) or neonatal spleen cell transplantation (Yunis, E. J., et al., 1974, Proc. Natl. Acad. Sci. U.S.A. 72:4100) as stem cell sources for hematopoietic reconstitution. Cells of neonatal thymus have also been transplanted in immune reconstitution experiments (Vickery, A. C., et al., 1983, J. Parasitol. 69(3):478-485; Hirokawa, K., et al., 1982, Clin. Immunol. Immunopathol. 22:297-304).
Freezing is destructive to most living cells. Upon cooling, as the external medium freezes, cells equilibrate by losing water, thus increasing intracellular solute concentration. Below about 10-15xc2x0 C., intracellular freezing will occur. Both intracellular freezing and solution effects are responsible for cell injury (Mazur, P., 1970, Science 168:939-949). It has been proposed that freezing destruction from extracellular ice is essentially a plasma membrane injury resulting from osmotic dehydration of the cell (Meryman, H. T., et al., 1977, Cryobiology 14:287-302).
Cryoprotective agents and optimal cooling rates can protect against cell injury. Cryoprotection by solute addition is thought to occur by two potential mechanisms: colligatively, by penetration into the cell, reducing the amount of ice formed; or kinetically, by decreasing the rate of water flow out of the cell in response to a decreased vapor pressure of external ice (Meryman, H. T., et al., 1977, Cryobiology 14:287-302). Different optimal cooling rates have been described for different cells. Various groups have looked at the effect of cooling velocity or cryopreservatives upon the survival or transplantation efficiency of frozen bone marrow cells or red blood cells (Lovelock, J. E. and Bishop, M. W. H., 1959, Nature 183:1394-1395; Ashwood-Smith, M. J., 1961, Nature 190:1204-1205; Rowe, A. W. and Rinfret, A. P., 1962, Blood 20:636; Rowe, A. W. and Fellig, J., 1962, Fed. Proc. 21:157; Rowe, A. W., 1966, Cryobiology 3(1):12-18; Lewis, J. P., et al., 1967, Transfusion 7(l):17-32; Rapatz, G., et al., 1968, Cryobiology 5(l):18-25; Mazur, P., 1970, Science 168:939-949; Mazur, P., 1977, Cryobiology 14:251-272; Rowe, A. W. and Lenny, L. L., 1983, Cryobiology 20:717; Stiff, P. J., et al., 1983, Cryobiology 20:17-24; Gorin, N. C., 1986, Clinics in Haematology 15(1):19-48).
The successful recovery of human bone marrow cells after long-term storage in liquid nitrogen has been described (1983, American Type Culture Collection, Quarterly Newsletter 3(4):1). In addition, stem cells in bone marrow were shown capable of withstanding cryopreservation and thawing without significant cell death, as demonstrated by the ability to form equal numbers of mixed myeloid-erythroid colonies in vitro both before and after freezing (Fabian, I., et al., 1982, Exp. Hematol. 10(l):119-122). The cryopreservation and thawing of human fetal liver cells (Zuckerman, A. J., et al., 1968, J. Clin. Pathol. (London) 21(l):109-110), fetal myocardial cells (Robinson, D. M. and Simpson, J. F., 1971, In Vitro 6(5):378), neonatal rat heart cells (Alink, G. M., et al., 1976, Cryobiology 13:295-304), and fetal rat pancreases (Kemp, J. A., et al., 1978, Transplantation 26(4):260-264) have also been reported.
Gene therapy refers to the transfer and stable insertion of new genetic information into cells for the therapeutic treatment of diseases or disorders. The foreign gene is transferred into a cell that proliferates to spread the new gene throughout the cell population. Thus stem cells, or pluripotent progenitor cells, are usually the target of gene transfer, since they are proliferative cells that produce various progeny lineages which will potentially express the foreign gene.
Most studies in gene therapy have focused on the use of hematopoietic stem cells. High efficiency gene transfer systems for hematopoietic progenitor cell transformation have been investigated for use (Morrow, J. F., 1976, Ann. N.Y. Acad. Sci. 265:13; Salzar, W., et al., 1981, in Organization and Expression of Globin Genes, A. R. Liss, Inc., New York, p. 313; Bernstein, A., 1985, in Genetic Engineering: Principles and Methods, Plenum Press, New York, p. 235; Dick, J. E., et al., 1986, Trends in Genetics 2:165). Reports on the development of viral vector systems indicate a higher efficiency of transformation than DNA-mediated gene transfer procedures (e.q., CaPO4 precipitation and DEAE dextran) and show the capability of integrating transferred genes stably in a wide variety of cell types. Recombinant retrovirus vectors have been widely used experimentally to transduce hematopoietic stem and progenitor cells. Genes that have been successfully expressed in mice after transfer by retrovirus vectors include human hypoxanthine phosphoribosyl transferase (Miller, A., et al., 1984, Science 255:630). Bacterial genes have also been transferred into mammalian cells, in the form of bacterial drug resistance gene transfers in experimental models. The transformation of hematopoietic progenitor cells to drug resistance by eukaryotic virus vectors, has been accomplished with recombinant retrovirus-based vector systems (Hock, R. A. and Miller, A. D., 1986, Nature 320:275-277; Joyner, A., et al., 1983, Nature 305:556-558; Williams, D. A., et al., 1984, Nature 310:476-480; Dick, J. E., et al., 1985, Cell 42:71-79); Keller, G., et al., 1985, Nature 318:149-154; Eglitis, M., et al., 1985, Science 230:1395-1398). Recently, adeno-associated virus vectors have been used successfully to transduce mammalian cell lines to neomycin resistance (Hermonat, P. L. and Muzyczka, N., 1984, supra; Tratschin, J.-D., et al., 1985, Mol. Cell. Biol. 5:3251). Other viral vector systems that have been investigated for use in gene transfer include papovaviruses and vaccinia viruses (see Cline, M. J., 1985, Pharmac. Ther. 29:69-92).
Other methods of gene transfer include microinjection, electroporation, liposomes, chromosome transfer, and transfection techniques (Cline, M. J., 1985, supra). Salser et al. used a calcium-precipitation transfection technique to transfer a methotrexate-resistant dihydrofolate reductase (DHFR) or the herpes simplex virus thymidine kinase gene, and a human globin gene into murine hematopoietic stem cells. In vivo expression of the DHFR and thymidine kinase genes in stem cell progeny was demonstrated (Salser, W., et al., 1981, in Organization and Expression of Globin Genes, Alan R. Liss, Inc., New York, pp. 313-334).
Gene therapy has also been investigated in murine models with the goal of enzyme replacement therapy. Thus, normal stem cells from a donor mouse have been used to reconstitute the hematopoietic cell system of mice lacking beta-glucuronidase (Yatziv, S., et al., 1982, J. Lab. Clin. Med. 90:792-797). Since a native gene was being supplied, no recombinant stem cells (or gene transfer techniques) were necessary.
The present invention is directed to hematopoietic stem and progenitor cells of neonatal or fetal blood, that are cryopreserved, and the therapeutic uses of such stem and progenitor cells upon thawing. In particular, the present invention relates to the therapeutic use of fetal or neonatal stem cells for hematopoietic (or immune) reconstitution. Hematopoietic reconstitution with the cells of the invention can be valuable in the treatment or prevention of various diseases and disorders such as anemias, malignancies, autoimmune disorders, and other immune dysfunctions and deficiencies. In another embodiment, fetal or neonatal hematopoietic stem and progenitor cells which contain a heterologous gene sequence can be used for hematopoietic reconstitution in gene therapy.
In a preferred embodiment of the invention, neonatal or fetal blood cells that have been cryopreserved and thawed can be used for autologous (self) reconstitution.
The invention also relates to methods of collection and cryopreservation of the neonatal and fetal stem and progenitor cells of the invention.
As used herein, the following abbreviations will have the meanings indicated: